The physiological correlates of the major stages in the morphogenesis of the mammalian central nervous system (CNS), including proliferation, apoptosis, migration and differentiation remain to be elucidated. We have used electrophysiological and digital videomicroscopic (DVM) techniques to study the development, differentiation and cellular distribution of physiological properties expressed by proliferating, progenitor and differentiating embryonic (E) rat CNS cells recorded in situ or in dissociated cell culture. Electrical recordings access excitable membrane properties in single cells and coupled pairs of cells in real time. DVM techniques that permit simultaneous study of cytoplasmic Ca (cCa) in approximately 100 cells at near real-time rates have been set up during FY97 and preliminary recordings carried out. Our long-term aim is to discover how excitable membrane properties together with fluctuations in cCa underlie the complex process of CNS morphogenesis. One principal line of investigation involves physiological co- differentiation of astrocytes and neurons in vitro. In vivo, astrocytes differentiate together with neurons throughout the late-embryonic mammalian CNS. During FY97, electrophysiological recordings of embryonic hippocampal neurons cultured directly on confluent astrocytes or in serum- free medium conditioned by astrocytes revealed that astrocytes promote the differentiation of specific neuronal properties, including membrane surface area and densities of inotropic current responses to major amino acid neurotransmitters and ligands (GABA, glycine, kainate and NMDA). We have not yet resolved whether the astrocyte-enhanced densities of the major transmitter receptor-coupled macroscopic currents reflects an increase in the surface expression of the receptor/channels and/or changes in their elementary biophysical properties. These astrocyte-mediated effects can be completely blocked by antagonism of either GABA receptor/Cl channels or inotropic glutamate (NMDA and non-NMDA) receptors, implicating each of these different families of receptors in the astrocyte-directed differentiation. The astrocyte effects can also be eliminated by preventing cCa levels to fluctuate in the differentiating neurons. Collectively, these results indicate that astrocytes secrete factor(s), which utilize GABA and glutamate receptors expressed on the neuronal surface, perhaps to generate fluctuations in cCa critical to the complex process of neuronal differentiation.